This invention relates to thyroid hormone receptors, and more particularly to non-nuclear effects of thyroid hormone.
Thyroid hormone (TH) has diverse effects on mammals, including effects on the neonatal and adult brain. In the developing animal, thyroid hormone regulates various events such as neuronal processing, glial cell proliferation, myelination, and neurotransmitter enzyme synthesis. The metabolically active form of thyroid hormone, 3,5,3xe2x80x2-triiodothyronine (T3), acts by binding to two receptors; TRxcex11 and TRxcex21. These two receptors are encoded by the c-erbAxcex1 locus. Two truncated receptor transcripts have been identified that are also transcribed from the c-erbAxcex1 locus; xcex94TRxcex11 and xcex94TRxcex12 (Chassande et al., 1997, Mol. Endocrinol. 11: 1278-1290). Neither truncated receptor has a DNA binding region and xcex94TRxcex11 has been shown to antagonize T3-induced transcriptional activation.
Type II iodothyronine 5xe2x80x2-deiodinase (D2) is the key enzyme in the pathway that mediates the conversion of intracellular thyroxine (T4) to 3,5,3xe2x80x2-triiodothyronine (T3). D2 concentration can be mediated by thyroid hormone concentration and is regulated by enzyme inactivation. The D2 activity appears to be more sensitive to T4 than T3. The degradation of the enzyme is energy-dependent and apparently requires the structural integrity of the actin cytoskeleton, i.e., is regulated at least in part by actin-based endocytosis. p29 is the substrate binding subunit of D2. T4 induces inactivation of D2 and initiates the binding of p29 to F-actin. The bound p29 is transported to an endosomal pool followed by dissociation of the F-actin-p29 complex (Farwell et al., 1993, J. Biol. Chem. 268: 5055-5062).
The present invention relates to the effects of thyroid hormone that are mediated by non-nuclear mechanisms. In particular, the invention relates to methods of identifying compounds that interact with a xcex94TRxcex11 polypeptide and/or xcex94TRxcex12 polypeptide (xcex94TRxcex11; xcex94TRxcex12). The invention also includes transgenic animals with altered or missing xcex94TRxcex11 and xcex94TRxcex12. Such animals are useful for identifying new targets for drug discovery.
Although xcex94TRxcex11 and xcex94TRxcex12 do not bind to 3,5,3xe2x80x2-triiodothyronine (T3), they do bind with high affinity to 3,3xe2x80x2,5xe2x80x2-triiodothyronine (reverse T3; rT3) and that binding can be displaced by thyroxine (T4) and rT3 (Example 2). The invention relates to the discovery that the association of myosin V with p29 vesicles is dependent on thyroid hormone, e.g., T4 and rT3, bound to a xcex94TRxcex12, and that stable complexes between p29, several synaptic vesicle proteins and myosin V can be isolated on actin fibers. Thus, T4, rT3, and certain analogs are useful for regulating actin-based endocytosis, especially movement of synaptic vesicles.
The invention features a method of assaying the functionality of a translation product of a mutant xcex94TRxcex12 gene in a cell. The method includes binding a labeled ligand for a xcex94TRxcex12 polypeptide to the translation product in a cell and measuring the amount, location, or rate of transit of the ligand in the cell. An increase in the amount, location, or rate of transit of the ligand in the cell compared to that in a cell that does not comprise a mutant xcex94TRxcex12 gene indicates an increase in functionality of the translation product. A decrease in the amount location, or rate of transit of the ligand in the cell compared to a cell that does not comprise a mutant xcex94TRxcex12 gene indicates a decrease in the functionality of the translation product. The ligand can be, e.g., a flavone, an aurone, or a T4 analog.
The invention includes an inhibitor of xcex94TRxcex12 expression or activity. The inhibitor can be, e.g., a flavone, an aurone, or a T4 analog.
The invention also features a method of identifying a candidate compound that modulates xcex94TRxcex12 activity by obtaining a xcex94TRxcex12 polypeptide, contacting the xcex94TRxcex12 with a test compound, assaying for binding of the test compound to xcex94TRxcex12, such that binding indicates that the test compound that binds to the xcex94TRxcex12 polypeptide is a candidate compound that modulates xcex94TRxcex12 activity. The test compound can be, e.g., a flavone, an aurone, or a T4 analog.
In another aspect, the invention provides a method of identifying a candidate compound that modulates xcex94TRxcex12 activity. This method includes obtaining a xcex94TRxcex12 polypeptide bound to a xcex94TRxcex12 ligand, contacting the xcex94TRxcex12 bound to the xcex94TRxcex12 ligand with a test compound, and measuring the displacement of the xcex94TRxcex12 ligand from the xcex94TRxcex12 polypeptide, such that displacement indicates that the a test compound is a candidate compound that modulates xcex94TRxcex12 activity. The test compound can be, e.g., a flavone, an aurone, or a T4 analog.
The invention also includes a method of identifying a candidate compound that modulates xcex94TRxcex12 activity. This method includes the steps of obtaining a test sample containing a xcex94TRxcex12, incubating the test sample with a test compound, and assaying the test sample containing the test compound for an alteration in type II 5xe2x80x2 deiodinase (D2) activity, such that a test compound that alters D2 activity when compared to a test sample that was not incubated with the test compound is a candidate compound. In this method, the test compound may decrease the amount of D2 activity. The test compound can be, e.g., a flavone, an aurone, or a T4 analog.
The invention also features a method of identifying a candidate compound that modulates xcex94TRxcex12 activity which includes the steps of obtaining a test sample containing a xcex94TRxcex12, performing an actin binding assay with the test sample in the presence of a test compound, such that a test compound that alters the binding of p29 vesicles to F-actin when compared to a test sample that was not incubated with the test compound is a candidate compound. The test compound can be, e.g., a flavone, an aurone, or a T4 analog.
The invention includes a compound identified by the any of the methods described above. The invention also includes an inhibitor of xcex94TRxcex12 expression or activity.
Other aspects of the invention are methods of treating a subject who has a neurologic disorder or a psychiatric disorder (e.g., a mood disorder or depression) by administering to the subject a therapeutically effective amount of a xcex94TRxcex12 ligand.
The invention also features an isolated nucleic acid molecule that includes a xcex94TRxcex12 targeting construct that contains a DNA sequence homologous to sequences encoding a mouse xcex94TRxcex12, such that when the construct is introduced into a non-human animal (e.g., a mouse) cell or an ancestor of the animal cell at an embryonic stage, and the construct-derived sequences are incorporated into an endogenous TRxcex1 gene, the cell does not express xcex94TRxcex12 in significant amounts (e.g., not more than 75%, 50%, 25%, 10%, or 5% of the level of expression in a cell or animal having a wild type gene). The invention includes a vector containing this nucleic acid. The construct can contain a nucleic acid sequence that is homologous to intron 7 of a mouse TRxcex1 gene or a nucleic acid sequence that is homologous to exon 10 of a mouse TRxcex1 DNA sequence. In some aspects of the invention, introduction of the construct into the cell disrupts the AP1, ctf, GR, SP1, or ets1 sequence of intron 7. The isolated nucleic acid molecule can also include a gene selection cassette.
The invention features a transgenic, non-human animal whose germ cells and somatic cells include a mutated TRxcex1 gene, the mutation being sufficient to inhibit binding of thyroxine (T4) to xcex94TRxcex12 transcribed from the gene. The mutated gene is introduced into the non-human animal or an ancestor of the animal at an embryonic stage, such that the animal, if homozygous for the mutation, has impaired motor function. The non-human animal can be a mouse, a rat, a goat, a sheep, or a pig. The invention includes a cell derived from the transgenic animal. The cell can be an astrocyte or other neuronal cell type. In such transgenic animals, the TRxcex1 gene can be mutated in intron 7 or in exon 10.
Another aspect of the invention features a transgenic non-human animal whose somatic and germ cells include a disrupted TRxcex1 gene, the disruption being sufficient to inhibit the binding of T4 to a xcex94TRxcex11 or xcex94TRxcex12 translation product of the TRxcex1 gene and the disrupted gene was introduced into the animal or an ancestor of the animal at an embryonic stage. Such an animal, if homozygous for the disrupted gene, has impaired motor function. The animal can be a rodent (e.g., a mouse or a rat), a goat, a pig, or a sheep. The disruption in such an animal can include a mutation in intron 7 or exon 10 of the TRxcex1 gene. The disruption can include a deletion of all or a part of intron 7 of the TRxcex1 gene or a deletion of all or part of exon 10 of the TRxcex1 gene.
A xe2x80x9ctransgenexe2x80x9d is any piece of DNA that is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from that cell or progeny of the organism. Such a transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene or part of a gene that is homologous with an endogenous gene of the organism.
A cell that is xe2x80x9ctransgenicxe2x80x9d is one which includes a DNA sequence inserted by artifice into a cell to become part of the genome of the organism which develops from that cell or progeny of the organism. As used herein, the transgenic organism can be an animal, generally a mammal (e.g., a rodent such as a mouse or rat), and the DNA (transgene) is inserted into the nuclear genome.
A xe2x80x9ctransgenic animalxe2x80x9d is an animal which includes a transgene. In general, the transgene is inserted into an embryonal cell and becomes a part of the genome of the animal which develops from that cell, or an offspring of such an animal. The transgene may introduce a heterologous DNA sequence into the embryonal cell or introduce an alteration such as a deletion, insertion, or substitution of an endogenous DNA sequence (e.g., by homologous recombination). In the transgenic animals described herein, the transgene causes cells to express an altered form of xcex94TRxcex11 or xcex94TRxcex12. Such animals include those produced using methods such as homologous recombination. In general, the animals produced by the transgenic technology of the invention are mammals although any animal that can be produced by such technology is encompassed by the invention. Mammals used for the invention include non-human primates, sheep, goats, horses, cattle, pigs, rabbits, and rodents such as guinea pigs, hamsters, rats, gerbils, and mice.
As used herein, a xe2x80x9chomologously recombinant animalxe2x80x9d is a non-human animal, e.g., a mammal, such as a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A xe2x80x9cxcex94TRxcex11 ligandxe2x80x9d or xe2x80x9cxcex94TRxcex12 ligandxe2x80x9d is a compound that binds to a xcex94TRxcex11 or xcex94TRxcex12, respectively. In some embodiments such a ligand binds to a xcex94TRxcex11 or xcex94TRxcex12 with an affinity of greater than or equal to 10xe2x88x928 Mol/L.
A molecule that xe2x80x9cspecifically bindsxe2x80x9d is a molecule that binds to a particular entity, e.g., a xcex94TRxcex11 or xcex94TRxcex12, but which does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample, which includes that particular entity, e.g., a xcex94TRxcex11 or xcex94TRxcex12.
The terms xcex94TRxcex11 xe2x80x9ccandidate compoundxe2x80x9d or xcex94TRxcex12 xe2x80x9ccandidate compoundxe2x80x9d refer to compounds that interact with or affect the activity of a xcex94TRxcex11 or xcex94TRxcex12. Such candidate compounds may be identified, e.g., by their ability to bind to one of the receptors, by their ability to displace a bound ligand from the receptor, by indirect assays such as ability to alter D2 activity when the candidate compound is incubated with the receptor in a D2-containing preparation, or by this ability to affect the association of myosin V with p29 vesicles. Candidate compounds may also be ligands.
A xe2x80x9ctest compoundxe2x80x9d is a compound used in the methods of the invention that is tested for its qualifications as a candidate compound.
A xe2x80x9chomologous sequencexe2x80x9d is a sequence with identity to a reference sequence. Calculations of homology (i.e., sequence identity) between sequences are performed as follows.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In an embodiment, the length of a reference sequence aligned for comparison purposes (e.g., when aligning a second sequence to a xcex94TRxcex11 or xcex94TRxcex12 amino acid sequence) is at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the length of the reference sequence. The length of the reference sequence can also be 100%. The reference sequence can be a full-length amino acid sequence of xcex94TRxcex11 or xcex94TRxcex12 or a partial sequence, e.g., a domain, intron, or exon (such as intron 7 or exon 10 of a mouse TRxcex1 sequence). In an embodiment, the length of a reference sequence aligned for comparison purposes (e.g., when aligning a second sequence to a xcex94TRxcex11 or xcex94TRxcex12 nucleic acid sequence) is at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the length of the reference sequence. The length of the reference nucleic acid sequence can also be 100%. The reference sequence can be a full-length nucleic acid sequence encoding a xcex94TRxcex11 or xcex94TRxcex12 or a partial sequence, e.g., a sequence that codes for a domain, intron, or exon such as intron 7 or exon 10. For some purposes, e.g., homologous recombination, the nucleic acid sequence may be a genomic sequence (e.g., include intron sequences). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid xe2x80x9cidentityxe2x80x9d is equivalent to amino acid or nucleic acid xe2x80x9chomologyxe2x80x9d). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Another set of parameters (e.g., that can be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences described herein can be used as a xe2x80x9cquery sequencexe2x80x9d to perform a search against public databases to, for example, identify other family members or related sequences (such as human xcex94TRxcex11, xcex94TRxcex12, or myosin V amino acid or nucleic acid sequences). Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules useful in the invention (such as human xcex94TRxcex11, xcex94TRxcex12, or myosin V). BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules useful in the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
The terms xe2x80x9csufficiently identicalxe2x80x9d or xe2x80x9csubstantially identicalxe2x80x9d are used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently or substantially identical.
A mutant xcex94TRxcex11 or xcex94TRxcex12 gene encodes a xcex94TRxcex11 or xcex94TRxcex12 polypeptide that includes a change in comparison to the wild-type amino acid sequence. In general, these changes arise from genetic engineering (e.g., by transgenic methods). These changes also include naturally occurring mutations and alleles.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Among the advantages of the present invention are new methods of identifying candidate compounds that affect the non-nuclear effects of thyroid hormone, e.g., those involving xcex94TRxcex11 and xcex94TRxcex12. Such compounds may be useful in treatments for disorders that involve such non-nuclear effects. The present invention also provides methods of treatment for disorders of the nervous system and psychiatric disorders such as depression, e.g., with compounds that bind to xcex94TRxcex11 and xcex94TRxcex12.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.